Micronutrients (vitamins and minerals) are not always present or available in sufficient amounts in foods. This may, inter alia, be due to specific deficits in the soil in which crops are grown, to a low bioavailability of the micronutrient, to unbalanced diets or to intestinal parasites. Recommended Nutrient Intakes (RNIs) or Recommended Daily Allowances (RDA) are aimed at informing the public about healthy levels of micronutrient intake.
It has been recognized that the intake of some nutrients at levels above the current RNIs may provide additional health benefits. Calcium intake levels above the RNIs are, for example, associated with increased bone mass in early adult life and in reduced risk of osteoporosis in later years.
Adequate calcium intake may be beneficial not only for the prevention and treatment of osteoporosis, but also for the reduction of risk of several diseases, including hypertension, colorectal cancer and oxalic renal calculi. Adolescents and the elderly are particularly vulnerable to the adverse effects of inadequate calcium intake. Recent studies and dietary recommendations have emphasized the importance of adequate calcium nutriture in pregnant women and in children, especially those undergoing the rapid growth and bone mineralization associated with pubertal development. The current dietary intake of calcium by children and adolescents in many parts of the world may well be below the recommended optimal levels.
While there is usually adequate calcium in the food supply to meet RNI levels, survey data indicate that many people are not consuming the recommended amounts of calcium. For example, many individuals do not consume milk products for a variety of health, cultural or personal reasons, and therefore have low intakes of calcium.
In order to alleviate micronutrient deficiencies, food fortification has become an essential element in nutrition strategies. An effective food fortification program improves the nutritional quality of a food supply by providing sources of essential nutrients and corrects inadequate intakes thereof.
Fortification, or enrichment, is defined as the addition of one or more vitamins or minerals to a food product irrespective of whether that food already contains that substance and concerns the vitamins A, B1, B2, B6, B12, C, D, E, K, niacin, folate, pantothenic acid and biotin and the minerals sodium, potassium, calcium, phosphorus, magnesium, iron, zinc, iodide, chloride, copper, fluoride, manganese, chromium, selenium, cobalt, molybdenum, tin, vanadium, silicon and nickel.
Food fortification is regulated by official governmental programs and is used as a tool for public health interventions (addition of iodine to salt), to restore nutrients lost during the processing of foods (addition of vitamin A to low fat or skim milk), to ensure the nutritional equivalence of substitute foods (addition of vitamin A to margarine) or to ensure the appropriate nutrient composition of special dietary foods such as meal replacements, nutritional supplements, low sodium foods, gluten-free foods, formulated liquid diets and sugar-free foods. Regulations determine which fortificants are allowable.
The presence of minerals like sodium, potassium, calcium and magnesium in the human diet is of eminent importance. Official documents state that for adults intakes of 1.1-3.3 gram/day sodium and 1.9-5.6 gram/day of potassium are estimated safe and adequate daily dietary intake levels. For calcium and magnesium, recommended daily allowances (RDA) are 0.8-1.2 gram/day and 0.3-0.4 gram/day, respectively. Sodium is generally present in sufficient levels and can easily be supplemented in the form of table salt. The situation for especially calcium is more complex.
In the human body calcium is primarily present in the bones and teeth (99%), and plays an important role in many enzymatic reactions in the intra- and extracellular fluids, blood clotting, muscle contraction, nerve transmission and in maintaining a regular heartbeat and blood pressure. Calcium is the most abundant mineral in the body and is predominantly present in the form of calcium phosphate. The bone acts as a large reservoir and buffer for Ca2− and PO43−. Plasma levels of Ca2− and PO43− are held within narrow limits by a parathyroid peptide called parathyroid hormone (PTH) which increases bone calcium mobilization and intestinal absorption and decreases renal excretion of calcium, and a second peptide of thyroid origin, calcitonin, which has an opposite action by decreasing bone calcium release and by increasing calcium and phosphorous excretion by the kidney. Vitamin A is also involved, mainly in the intestinal absorption of calcium.
Daily losses of calcium must be replaced through dietary intake. Recognized sources of calcium are food sources (naturally occurring and/or as food fortificants), mineral supplements and miscellaneous sources such as antacids (for the treatment of gastric ulcers and acid reflux). The largest source of dietary calcium for most persons is milk (1.2-1.4 g calcium/liter) and other dairy products. Other sources of calcium are, however, important, especially for achieving calcium intakes of 1.2-1.5 g/day (US RNI). Most vegetables contain calcium, although at low density. Therefore, relatively large servings are needed to equal the total intake achieved with typical servings of dairy products.
Calcium adequacy depends not only on the quantity of intake, but also on the portion of calcium in food which can be absorbed through the intestines and is useable in the body, called bioavailability. Even if the daily food intake contains sufficient calcium to theoretically keep the balance of calcium in the body adequate, a lack of calcium in the body may occur. The bioavailability of calcium from vegetables is generally high. An exception is spinach, which is high in oxalate, making its calcium virtually non-bioavailable. Some high-phytate foods, such as whole bran cereals, may also have poorly bioavailable calcium.
While vitamin D and the parathyroid hormone are involved in the active intestinal absorption process, phosphates play a major role in the passive absorption based on diffusion. This absorption process is positively stimulated by the concentration of dissolved calcium: the greater the solubility of the calcium compound, the stronger the passive absorption.
Almost all calcium ingested and present in the stomach is essentially in ionic form because of the low pH conditions. However, when calcium enters the intestine the conditions are more neutral (pH 6-7 and the calcium may precipitate as insoluble calcium phosphate, depending upon the amount of phosphate present. The body is unable to absorb these precipitated calcium phosphates. Precipitation is most likely to occur when the calcium:phosphate ratio in the food or in the intestine is below 1. In Europe this ratio is around 0.6 and in the US this figure is even lower. Therefore, calcium supplementation of foods is beneficial for numerous reasons.
Several products have been introduced that are fortified with calcium. These products, most notably orange juice, are fortified to achieve a calcium concentration similar to that of milk. Limited studies suggest that the bioavailability of the calcium in these products is at least comparable to that of milk. The data on bioavailability of the various calcium fortificants are not conclusive. Several studies indicate equal bioavailability between calcium carbonate, calcium sulphate, calcium citrate and calcium lactate whereas other investigations show that organic calcium salts may possess higher bioavailability than inorganic salts such as calcium carbonate. Generally, bioavailability is determined by the solubility of a calcium compound as mentioned previously. Solubility is defined as the soluble concentration of a certain compound in water in the presence of the crystalline form of the compound, i.e. soluble and crystalline forms are in thermodynamic equilibrium.
The major problems involved in fortifying foods include the identification of suitable vehicles, selection of appropriate fortificant compounds, determination of technologies to be used in the production of the fortificant and in the fortification process and the influence on taste and consumer satisfaction of the food product. The selection of the appropriate calcium source for a specific fortification application is usually based on the consideration of a number of properties associated with the respective product such as taste, calcium content, bioavailability, absorbability (usually 10-40%), bioavailability and, most importantly in the case of calcium, solubility, which determines bioavailability and absorbability. Economic considerations are, evidently, other important factors. A calcium fortificant ideally exhibits a solubility in excess of 3 grams of Ca2+ per liter.
Calcium carbonate, from oyster shells or limestone, is the most widely used calcium supplement because of its high calcium content and low cost. However, calcium carbonate has the disadvantage of developing CO2 in the stomach, tends to produce a chalky mouthfeel, may impart a bitter, soapy or lemony taste on the finished product and may produce sediment in the food product. Most importantly, calcium carbonate is poorly soluble (<0.1 g/L at 25° C.) and therefore has a low absorption. Calcium phosphates, such as dicalcium phosphate impart a gritty mouthfeel, have a bland flavor and are also poorly soluble (<0.1 g/L at 25° C.).
Some organic calcium salts like calcium lactate, and calcium gluconate exhibit higher solubility and are better absorbed by the body. Tricalcium citrate offers a high calcium content and a neutral taste but is the least soluble (0.2 g/L at 25° C.). Calcium lactate, on the other hand, is highly soluble (9.3 g/L at 25° C.) making it very beneficial for obtaining a high calcium content in a food product. However, calcium lactate is perceived as bitter. Calcium gluconate is somewhat less soluble (3.5 g/L at 25° C.) as calcium lactate and it is considered to be one of the most neutral calcium salts with respect to taste allowing for high levels of addition in food products without negative impact on taste. Calcium gluconate is also very compatible with the human body. It does essentially not show any toxicity or astringency, and is therefore well tolerated.
Calcium gluconate is the calcium salt of gluconic acid. In order for the product to be registered as a food fortificant, it must be prepared at high purity, i.e without the presence of by-products that are unregistered. An important disadvantage of calcium gluconate is that it is relatively costly to produce. Several processes for the production of gluconates are described in the literature. U.S. Pat. No. 4,845,208 describes a process for the production of aldonic acids by aldose oxidation using a palladium-based catalyst. The disadvantage of this method is the use of very expensive and toxic catalysts and the formation of several by-products.
U.S. Pat. No. 5,102,795 describes the electrochemical oxidation of glucose to gluconic acid using sodium bromide and a neutralizing base. This method is presently used for production of gluconates but suffers from the problem that sodium bromide or bromide has to be separated from the final product.
A production process that generates gluconates of high purity (without the formation of by-products in the reaction mixture) is described in the international patent application 96/35800. In that publication, a combination of glucose oxidase and catalase enzymes is used to enzymatically convert glucose to gluconic acid while neutralizing with sodium hydroxide to obtain sodium gluconate at a yield close to 100% and practically without impurities. Sodium gluconate is highly soluble (380 g/L at 20° C.) and the inventors of that international patent application were able to achieve complete conversion of 273 g/L of glucose into gluconic acid (in the form of liquid sodium gluconate). Although the sodium ions may be exchanged by calcium ions to obtain calcium gluconate, such a process is impractical because of problems of calcium precipitation and the fact that a monovalent ion has to be exchanged for a divalent ion, attached to the resin, and industrial production of calcium gluconate in this way is unattractive. Moreover, ion exchange is an expensive unit operation and the process generates waste salts.
International patent application 97/24454 describes the enzymatic conversion of glucose into gluconic acid (in the form of liquid sodium gluconate) up to initial glucose concentrations of 450 g/L at a pH of 6,0 by using a pressurized reactor system and glucose oxidase from Aspergillus niger and catalase from Micrococcus luteus. However, this process does not allow for high yield productions of calcium gluconate since the solubility of calcium gluconate (30 g/L at 20° C.) is an order of magnitude lower than that of the corresponding sodium salt.
Also, calcium gluconate can be prepared by microbial fermentation as described by Shah and Kothari (Biotechnol. Lett. 1993; 15:35-40) and by Klewicki and Krol (Pol. J. Food Nutr. Sci. 1999; 8:71-79). The drawback of those procedures is related with the need to purify the calcium gluconate post-production from by-products formed and to separate the micro-organisms from the product.
For the production of calcium gluconate an enzymatic process is preferred because of its high specificity and the resulting high purity of the product. However, the solubility of calcium gluconate under the conditions required for optimal enzymatic conversion (pH 5-7, 30-35° C.) is only 40 g/L. In order to obtain industrial quantities of the product, large reactors are needed and the concentration of the calcium gluconate crystals requires the evaporation of large amounts of water. Increase of the initial glucose concentration and instant crystallization of the formed calcium gluconate results in an increased viscosity of the reaction mixture and a concomitant decrease in the rates of oxygen transfer and enzymatic conversion. Intensification of agitation to restore oxygen transfer and overcome viscosity problems, e.g. by using Rushton turbines for stirring will raise equipment costs and heightens the risk of damage to the used equipment and concomitant metal contamination (chromium or nickel) of the final product. These drawbacks have rendered the enzymatic production of calcium gluconate on an industrial scale unattractive.